Summary With no single effective therapy for either the prevention or treatment of bronchopulmonary dysplasia (BPD), a chronic lung disease of preterm infants, the need for new tools to treat and reduce risk of further complication is urgent. Mesenchymal stem/stromal cell (MSC) therapy has shown promise in preclinical models of BPD, demonstrating both histological and functional benefits. We have shown that the therapeutic capacity of MSCs is comprised in their secretome, and that the major therapeutic vector therein is represented by the exosomes. Exosomes are submicron vesicles that harbor a diverse array of bioactive cargo (e.g. lipids, diverse proteins, and small non-coding RNAs). Recently, using a murine model of hyperoxia-induced BPD, we demonstrated that a bolus dose of human MSC-exosomes (termed MEx), significantly improved lung morphology, pulmonary development and lung function, decreased lung fibrosis, restored pulmonary blood vessel loss and ameliorated pulmonary vascular remodeling and pulmonary hypertension. We have also demonstrated that MEx are readily taken up by macrophages (M?) both in vitro and in vivo and, as a result, shift the M? phenotype to an anti- inflammatory, anti-fibrotic, and pro-regulatory state. However, despite the promising therapeutic potential of MEx, our understanding of their bioactive properties and the molecular mechanism(s) responsible for such effects remain unclear. Exosomes are a heterogeneous EV population. Exosome subpopulations are known to differ in biophysical, proteomic and RNA repertoire. Consequently, different MEx subsets mediate alternative biological functions. In this proposal we predict that only a certain subtype of MEx is responsible for the therapeutic effects, and that this ?bioactive? MEx subset dampens inflammatory signaling in the lung via modulation of M? phenotype and prevents the development of hyperoxia?induced vascular and alveolar injury. To test these hypotheses, we propose the following specific aims (SA). SA#1: To isolate, characterize and define therapeutic (?bioactive?) MEx subsets. SA#2: To assess MEx in vivo biodistribution and anti-inflammatory/immunomodulatory capacity. SA#3: Investigate how MEx impacts the epigenetic landscape of target cells in our experimental BPD model. Collectively, this proposal will provide important insights in MEx biology and MEx-target cell interaction, that can be leveraged to develop effective, novel therapeutic modalities for BPD.